The ultimate goal of organ transplantation is to achieve sustained allograft acceptance with minimal broad-based immunosuppression and off-target toxicity. Unfortunately, current approaches using corticosteroids (CCS) and calcineurin inhibitors (CNIs) have substantial nephrotoxicity. Our Co-Investigator, Dr. Woodle has shown that when combined with T cell- depleting induction, belatacept, a biologic that inhibits T cell co-stimulation, provides immunosuppression that is similar to CNI/CCS-based regimens without toxicity. However, the remaining rejection under belatacept is more difficult to control and requires further understanding. To this end, we have developed the ability to analyze rejection using single cell RNA sequencing of cells isolated from tissue biopsies. Further, our novel approach will also specifically identify the allo-reactive cells in the rejecting allograft. This will be done by performing a functional mixed lymphocyte reaction (MLR) assay using PBMCs and identify the T cell receptor complementarity determining region (CDR) of those cells responding in the MLR assay. These sequences will serve as a fingerprint to identify CDRs from T cells in the allograft of the same patient using a novel kit from 10x genomics, which combines analysis of CDR3 regions and whole cell transcriptomes. We hypothesize that (i) the phenotype of donor reactive CD8+ T cells will differ in PBMCs from belatacept-refractory (CD28-CD38+) vs CNI/CCS-refractory (CD28+CD38+) patients; (ii) MLRs from peripheral blood cells can identify donor-specific T cell receptors and their unique CDR3 sequences can be used to identify donor-specific T cells in the rejecting allograft; and (iii) the transcriptomes and underlying biology of donor- specific T cells from patients rejecting under belatacept will be distinct from those rejecting under CNI/CCS-based regimens. To test these hypotheses, we Aim to (1) determine the transcriptomes of CD8+ T cells in kidney biopsies during rejection under belatacept versus CNI/CCS-based regimens, and (2) identify the donor reactive CD8+ T cells in the PBMCs of belatacept vs CNI/CCS-refractory patients, determine their TCR? and ? CDR3 sequences, and match them with CDR3 sequences from the graft.